Dai Zhenyu,1 Chen Jing,1 Xu Qun,1 and Jeffrey Rohrer2

1
Thermo Fisher Scientific, Shanghai, People’s Republic of China;
2
Thermo Fisher Scientific, Sunnyvale, CA, USA

Appli cat i on N ote 1 0 6 9

Two-Dimensional HPLC Determination
of Water-Soluble Vitamins in a
Nutritional Drink

Key Words
Food Analysis, Food Quality, Acclaim PolarAdvantage Column,
Acclaim 120 C18 Column, Hypersil GOLD Phenyl Column

Goal
To develop an efficient high-performance liquid chromatography
(HPLC) method for simple and sensitive determination of watersoluble vitamins in a complex multivitamin/mineral drink. Target
analytes are B group vitamins, including thiamine (VB1), riboflavin
(VB2), nicotinamide (VB3), pantothenic acid (VB5), pyridoxine (VB6),
biotin (VB7), and cyanocobalamin (VB12); and ascorbic acid (VC).

Introduction
Vitamins are a well-known group of compounds that are
essential for human health. They can be classified into two
main groups: water- and fat-soluble. With the exception of
VB6 and VB12, water-soluble vitamins are not stored in the
body. Thus, if one’s dietary vitamin intake is insufficient,
a vitamin supplement should be added to the diet.
The vitamin supplement can be in tablet form, a clear
vitamin-enhanced functional drink, vitamin-enhanced
milk, or a nontransparent multivitamin/mineral nutritional
drink with additions of other substances (e.g., fruit
extracts) that make it more complex than clear products.
To ensure that these products contain the labeled amounts
of vitamins, a number of reliable quality control assays are
available.1-3 For a vitamin tablet or a clear functional
drink, the analysis is relatively simple and a routine
HPLC method (e.g., a C18 column with UV detection)
is satisfactory for quantifying the vitamins.4
Some samples, however, have too many additional components to allow a routine HPLC vitamin-quantification
method. Vitamin-enhanced milk and a nontransparent
multivitamin/mineral nutritional drink—referred to as a
multivitamin nutritional drink throughout the rest of this
study—are two such samples. In addition to vitamins,
these samples also supply amino acids, minerals, coenzyme
Q10, the compounds contained in grape extracts, and
more. These additional compounds interfere with the
separation of vitamins, making quantification difficult.

Therefore, a simple and sensitive two-dimensional HPLC
(2D-HPLC) method is needed to quantify vitamins in these
complex samples.

Equipment and Software
• Thermo Scientific™ Dionex™ UltiMate™ 3000 x2 Dual
HPLC system, including:
− DGP-3600RS Dual-Gradient Rapid Separation
Pump System with SRD-3600 Integrated Solvent
and Degasser Rack
− WPS-3000TRS Rapid Separation Wellplate Sampler,
Thermostatted, with a 100 μL sample loop and a
100 μL syringe
− TCC-3000RS Rapid Separation Thermostatted
Column Compartment equipped with two
2p–6p valves
− DAD-3000RS Rapid Separation Diode Array
Detector with 13 µL flow cell
− Mixer for 800 μL Mixing Volume (P/N 6040.5750)
• Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System (CDS) software version 7.1
• Thermo Scientific™ Orion™ 2-Star pH Benchtop Meter

China: – VB1 – VB3 – VB6 – VB12 – VB2 – VB5 – VB7 – VC For the preparation of mixed working standard solutions for calibration.0 10 VB2 0.15 0. add the appropriate volume of the mixed stock standard solution into 10 mL glass vials and bring to 10 mL with DI water. VB6. Also. VB12. and the concentration of the other vitamins will be 50 μg/mL. Make a fresh preparation of VC. VB12. the concentrations of VB5 and VB7 will be 100 μg/mL. VB12.2 MΩ-cm resistivity • Acetonitrile (CH3CN) for HPLC (Fisher Scientific P/N AC610010040) • Potassium Phosphate Monobasic (KH2PO4) (Fisher Scientific P/N P286-1) • o-Phosphoric Acid (H3PO4). and VC stock solutions to a 1 mL sample vial. because of the limited solubility of VB2 in water. To prepare water-soluble vitamin standards of VB1. gluconate) * Serving size: 1 fluid oz Amount Per Serving* 9g 10000 IU 1000 mg 200 IU 200 IU 300 mcg 30 mg 30 mg 30 mg 150 mg 30 mg 300 mcg 500 mcg 400 mcg 600 mg 80 mg 4 mg 300 mg Ingredient Amount Per Serving Zinc (as gluconate) Selenium (as L-selenium methione) Copper (as gluconate) Manganese (as gluconate) Chromium (as amino acid chelate) Potassium (as citrate) Choline (as bitartrate) Inositol Boron (as amino acid chelate) Amino Acid Complex (proprietary formula) Grape Seed Extract Coenzyme Q-10 Dimethyl Glycine Paba Citrus Bioflavonoids Glucolactone Plant-Derived Minerals 15 mg 100 mcg 1 mg 5 mg 200 mcg 100 mg 30 mg 30 mg 1 mg 125 mg 25 mg 5 mg 25 mg 30 mg 13 mg 150 mg 600 mg . Beijing. the concentration of VB2 will be 15 μg/mL. Mixed Working Standard Solution Volume of Mixed Stock Standard Solution for a 10 mL Preparation (mL) Concentration of Each Vitamin (mg/L) 1 2 3 4 5 6 0. VB5. Dilute the drink sample with DI water if necessary and filter through a 0.0 15 VB5. Sample Preparation Preparation of Standard Solutions A multivitamin nutritional drink supplemented with VB1.0 10 5. If a 10 mL volumetric flask were used.2 1. 0. Table 2 lists the sample components.0 VB1.1 0. amino acid complex. Bring the final volume to 1 mL by adding 50 μL of DI water to make the mixed stock standard solution.5 3. 85%. VB7. See Table 1 for details. VB6. Table 1. Labeled values of the multivitamin nutritional drink. 18. VC 0. but that would exceed the precision range of the balance.2 1. VB2.45 µm filter. VB3. VB6. Preparation of mixed working standard solutions of water-soluble vitamins. Reagents and Standards • Deionized (DI) water.5 1. VB7 0. VB3. The drink also contained some fat-soluble vitamins.3 mg of VB2 would have to be weighed.1 0.03 0. VB6. coenzyme Q10. 100 μL each of VB5 and VB7 stock solutions. (Fisher Scientific P/N A260-500) • Products from the National Institute for the Control of Pharmaceutical and Biological Products.0 2. VB3. VB12.03 mg/mL. compare the labeled values to the calibration ranges in this study.2 Add 500 μL of VB2 stock solution. Store the sample in a brown bottle at 4 °C before analysis. VB7. Ingredient Total Carbohydrate Vitamin A Vitamin C Vitamin D3 Vitamin E Vitamin K1 Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B5 Vitamin B6 Vitamin B7 Vitamin B12 Folate Calcium Phosphorus Iron (as gluconate) Magnesium (as citrate. grape seed extract.0 2. weigh 10 mg of the vitamin powder and add DI water to 10 mL in a volumetric flask to make stock solutions of 1. and VC was provided by a customer. and 50 μL each of VB1. In this mixed stock standard solution. and other ingredients added to meet daily nutritional needs. due to its limited stability. Weigh 3 mg of VB2 into 100 mL DI water to address the solubility issue.3 1.0 20 100 10 50 Table 2. To determine if the sample needs to be diluted. VB5.0 mg/mL for each vitamin. minerals. decrease the concentration of the VB2 stock solution to 0. VB3. and VC.02 0.

0 80. Conventional HPLC Method for the Determination of Vitamins Currently. pH 3.8 mL/min Inj.0 with H3PO4) Second Dimension (Left Pump) B.5 Due to the irreversible impact of the ion-pairing agent on column performance.8 0.5 6_1 1_2 6_1 1_2 6_1 1_2 6_1 1_2 6_1 1_2 6_1 1_2 6_1 1_2 6_1 6_1 100 100 50 0 0 — — — — — — — — — — — Left Valve Position Time (min) Flow Rate (mL/min) 1_2 6_1 1_2 6_1 1_2 0 5 12.4 g KH2PO4 in 1 L water and adjust the pH to 3. Volume: 10 μL Temperature: 25 °C Detection: UV. dimethyl sulfoxide.0 100 100 — — — — — — — — — Results and Discussion Conditions First Dimension Columns: For water-soluble vitamins except for VB12.8 0.6 × 150 mm (P/N 059148) Mobile Phase: Same as used in the First Dimension Flow Rate: 0.0 21. 5 μm Analytical. First Dimension (Right Pump) Time (min) Flow Rate (mL/min) 0 14 23. 4.60 15.8 — — — — — — — — — 1_2 6_1 1_2 6_1 1_2 6_1 1_2 6_1 1_2 6_1 1_2 %A (25 mM Phosphate Buffer.8 0. absorbance at 210 and 254 nm Second Dimension Column: Acclaim 120 C18. 4.e. Thermo Scientific™ Acclaim™ PolarAdvantage™ (PA). 268. Pharmacopeia (USP) method for the separation of a mixture of all eight water-soluble vitamins.0) 100 100 65..5 30 — — — — — — — — — 0.0 21.8 — — — — — — — — — — — %A (25 mM Phosphate Buffer. %B (CH3CN) 0 0 35.82 15.5 24 30 — — — — — — — — — — — 0.12 13.28 14.12 28. 5 μm Analytical.35 13. extensive research was conducted to search for methods without ion-pairing agents.8 0.3 Table 3. pH 3. Thermo Scientific™ Hypersil GOLD™ Phenyl Analytical HPLC. 25 mM phosphate buffer (dissolve ~3. 4.8 0.97 16. 5 μm.S.6 × 250 mm (P/N 061321) For VB12.0 80.0 20.8 0.0) Valve Switching %B (CH3CN) Time (min) Right Valve Position 0 0 50 0 0 — — — — — — — — — — — 0 3.89 7. CH3CN Gradient: See Table 3 Flow Rate: 0. sodium perchlorate. phosphoric acid. and water are needed for biotin determination) and involves an ion-pairing agent to retain hydrophilic vitamins.8 8.8 0. acetonitrile.0 20.61 14.3 5.8 0. 245.8 0. there is no U.5 13.8 0. A USP method for individual vitamins is complicated (i. and 291 nm These conditions apply to Figures 3 through 7. Gradient program and valve switching. and this for a simpler mobile phase.0 0 0 — — — — — — — — — .32 5.94 4.42 15.8 mL/min Temperature: 25 °C Detection: UV absorbance at 210.6 × 150 mm (P/N 25905-154630) Mobile Phase: A.

the water mobile phase in the second dimension will dilute the acetonitrile from the first-dimension mobile phase. Use these values to switch the valves. Not only do two columns with different chemistries provide additional separation power. 0%. The in-line UV detector determines where the water-soluble vitamins elute. switch the left valve to the 1_6 position so that these vitamin fractions Column: Acclaim PA1. VB5 100 6. the vitamin peaks cannot be precisely quantified. Obviously. 25 mM phosphate buffer (dissolve ~3. Volume: 10 µL Temperature: 25 °C Detection: UV absorbance at 210 nm Samples: A. Switch the right valve to the 1_2 position to individually transfer the vitamin peaks to the flow path of the second column. Dual Gradient Pump Column 2 Minutes 8 are directly transferred to the second column. 4. CH3CN Gradient: CH3CN. UV Detector 1 will be connected between the two columns. VB1 2. 0–14 min. Peaks 1–3 cannot be quantified.5–24 min. switch the left valve to the 1_2 position to put the 750 μL mixer in line. For late eluting vitamin peaks.6 × 250 mm Mobile Phase: A. Column 1 Waste 5 2 Autosampler L 4 1 A Dual Gradient Pump Mixer 5. 23. Standard mixture B. the additional supplements can interfere with vitamin separation and. VB6 50 mg/L 50 50 50 500 mAU 6 B 3 0 –50 0 5 Waste Figure 2. VC 3. choose the proper particle size and length of the seconddimension column to keep the backpressure below the pressure limit of the flow cell. R Detector 2 10 7 Figure 1. An injection of the mixed standard determines the start and end times for each vitamin peak. ultimately. Figure 2 shows the configuration of the 2D-HPLC system used for this study. Peaks 5–8 can be detected in the sample. VB3 4. The pressure limit command in the instrument method can also be set to stop the flow in any situation when backpressure may become too high. However.4 g KH2PO4 in 1 L water and adjust pH to 3. Figure 1 shows that use of a typical HPLC method for a multivitamin nutritional drink results in the water-soluble vitamin peaks being hard or even impossible to quantify due to the large number of UV-absorbing interfering peaks. This enables the second-dimension column to trap the vitamin peaks. the backpressure of the second-dimension column may exceed the pressure limit of the UV Detector 1 flow cell. then dilute with DI water if necessary). acidic or neutral phosphate buffer/organic solvent mobile phases have been used to separate vitamins in an extract of a multivitamin tablet and in vitaminenhanced functional drinks that are less complex than the one investigated here. Multivitamin nutritional drink Peaks: 1. their detection by UV absorbance.8 mL/min Inj. The 2D-HPLC Method Two-dimensional HPLC has been used to achieve efficient separation of complex samples. Thus. VB7 100 . Comparison of the UV spectra of the standard and the sample confirmed that Peaks 5–8 were not pure enough for quantification. but use of heart-cutting technology in on-line 2D-HPLC also simplifies the separation in the second dimension.0 with H3PO4) B. In this configuration. After simple sample preparation (filter the sample. 5 µm Analytical.1 For complex samples such as certain multivitamin nutritional drinks. VB12 50 7. For early eluting vitamin peaks. Configuration of the 2D-HPLC system. A standard mixture (A) and a multivitamin nutritional drink sample (B) using a conventional HPLC method.5 min. but due to all the additional peaks. 0–50%. VB2 15 8. 15 20 25 Note: When the right valve is switched to the 1_2 position. 14–23. The work shown here uses 2D chromatography to determine water-soluble vitamins in a complex sample. 50–0% Flow Rate: 0. inject the sample into the first dimension and partially separate it using an Acclaim PA column.4 Recently.

VB1 2. It worked well for late eluting peaks. The traditional approach to this problem is to provide water to dilute the acetonitrile in the transferred fraction with a tertiary pump so the fraction can be retained on the trap column ahead of the second column. but the early eluting peaks broadened (Figure 3. in-line for Peaks 4–8 Peaks: 1. a 750 μL mixer was configured in line before the eluted fraction was transferred to the second dimension. and 8 disappear when they are directly transferred to the second dimension. 750 µL mixer off-line for Peaks 1–3. Choice of wavelength for the UV detector Vitamins B1. VB12 50 7. which are the wavelengths of maximum UV absorbance of each. whereas late eluting peaks are transferred into the 750 μL mixer before the second column.Development of the 2D-HPLC Method Valve switching To achieve optimal resolution and peak shape in 2D-HPLC applications. Thermo Scientific Application Note (AN) 1023 demonstrated an alternative way to solve this problem. VB2 15 8. Chromatogram A shows that Peaks 5. B6. even with a 100% water mobile phase. Ideally. . the critical part of method development is the transfer of first-dimension peaks to the second-dimension column. VB3 4. respectively. VB6 300 50 mg/L 50 50 50 5. 268. 5 Detection: UV absorbance at 210 nm Valve Position: See Table 3 Configurations: A. These peaks cannot be trapped at the head of the second column. Obviously. Thus. Figure 3. Second-dimension analysis of the standard mixture with three different configurations. the two mobile phases usually will have different concentrations of acetonitrile or other organic solvent. Figure 3. which is close to its maximum UV absorbance. to minimize noise. VB5 100 6. the late eluting peaks from the first column are contained in too high a concentration of acetonitrile to be retained on the second column.6 However. 291. both were detected at 210 nm. VC 3. 7. Vitamin B12 was detected with the 245 nm channel. B2. the configuration was modified so that early eluting peaks are directly transferred to the second column. VB7 100 A 4 mAU 6 3 1 2 –50 200 6 B 4 mAU 7 5 –50 250 4 C 6 3 mAU 1 5 2 –50 8 0 5 10 15 Minutes 20 7 8 25 30 Figure 3. the need for an additional pump limits this application.7 Briefly. therefore. In this study. Vitamin B3 was detected at 268 nm. If desired. the first-dimension mobile phase being cut and transferred with target peaks to the second dimension will be same as the seconddimension mobile phase at the time of transfer. and C were detected at 245. After conducting additional experiments. But in reality. the authors discovered that early eluting vitamin peaks broadened due to their physiochemical characteristics. which had high concentrations of acetonitrile. Then the peak mobile phase was mixed extensively with the second-dimension starting mobile phase (water phase) before the second-dimension separation. The maximum UV absorbance wavelengths of VB5 and VB7 are below 200 nm. a fifth channel of 361 nm can be configured to detect VB12 at its absorbance maximum. and 245 nm. 750 µL mixer always in-line C. the authors initially applied the AN 1023 approach for all target peaks. Chromatogram B). 750 µL mixer always off-line B. Chromatogram C shows that the final configuration works well for all eight water-soluble vitamins.

VB1 50 mg/L 2. Detection: UV absorbance at 245 nm Valve Position: See Table 3 Samples: A. A VB12 standard (A) and a multivitamin nutritional drink sample (B) using a Hypersil GOLD Phenyl column in the first dimension.8 Most water-soluble vitamins can be separated with good resolution using this column combination. A standard mixture (A) and a multivitamin nutritional drink sample (B) using an Acclaim PA column in the first dimension. VB2 15 1. Figure 4 shows that VB12 was not detected at 245 nm when an Acclaim PA column was used in the first dimension. VB12 standard mixture (20 mg/L) B. VC 50 3. VB12 50 200 5 6 mAU mAU 1 B 1 A –25 B 1 2 5 3 6 A 0 5 10 15 21 22 Minutes 23 24 25 Figure 5. making quantification of VB12 impossible.6 Column choice and its impact on separation Several combinations of columns were tested. 4 –50 20 20 25 30 Minutes Figure 4. However. VB3 4. VB12 50 6. The authenticity of the VB12 peak was confirmed by its UV spectrum. VB6 The authors searched for another column to use for the first dimension. Multivitamin nutritional drink sample Detection: UV absorbance at 245 nm Valve Position: See Table 3 Samples: A. but VB12 coelutes with an impurity in the drink sample. 50 50 Peak: 5. Multivitamin nutritional drink Peaks: 1. VB12 was detected (Figure 5). Previous work suggested that good choices for most water-soluble vitamins are an Acclaim PA column used as the firstdimension column and an Acclaim 120 C18 column used as the second-dimension column. Standard mixture B. The Hypersil GOLD Phenyl column worked well for this purpose. . when the Hypersil GOLD Phenyl column was used in the first dimension.

Vitamin B3 was surprisingly not detected. Water-Soluble Vitamin Retention Time RSD Peak Area RSD VB1 0.5–50 A=0. the method is capable of determining VB3.0153 0. therefore. The other water-soluble vitamins were detected close to their labeled values. reproducibility was estimated by making eight replicate injections of water-soluble vitamins.02 0.20 VB2 268 0.9999 0.08 VB2 0. An overlay of the eight injections is shown in Figure 6.32 VB3 0.23 0.1237 0.0276 0.9618c-0.1755c-0.02 0.62 VB5 0. VB7 100 4 3 1 5 2 H F E D C B A Calibration linearity for the water-soluble vitamins was investigated by making three replicate injections of a mixed standard prepared at five or six different concentrations.5–50 A=0. Linearity.03 0. VB3 4. Calibration data and MDLs for the water-soluble vitamins.9978 0.11 0. Reproducibility of retention time and peak area for water-soluble vitamins. VC 3. and Detection Limits Prior to sample analysis. –40 0 5 10 15 Minutes VB1 245 0.45 VB7 0.48 VC 0. Range (µg/mL) 7 8 G Reproducibility. Detection Wavelength (nm) 6 mAU 3. These data are reported in Table 5.9995 0.1080 0.87 VB12 0.18 Regression Equation VB5 210 1.0423 0.03 0.Table 4.5–10 VB3 268 VB6 291 20 25 30 Figure 6. The authors found linear calibration curves for each vitamin over the ranges evaluated.0101c 0.5 VC 245 — — — — . Table 5. The RSDs for retention time and peak area are shown in Table 4.9999 0.1 Peaks: 100 1. Chromatogram overlays of eight consecutive injections of a mixture of water-soluble vitamin standards. consecutive injections 1–8 r MDL (µg/mL) A=0. The external standard method was used to calculate the calibration curve and quantify these compounds in samples.2385c+0.03 VB7 210 2.15–15 A=0. Sample Analysis Vitamins B7 and B12 were not detected in the multivitamin nutritional drink.02 0.30 0. VB5 100 6.0161 0.1026c+0.0–100 A=0.05 1.2918c+0.9999 0. VB1 2. VB6 50 mg/L 50 50 50 5.36 VB6 0.0462c+0. possibly due to their low concentration in the sample. The singlesided Student’s t test method was used for estimating method detection limits (MDL).5–50 A=0.9999 0. VB2 15 8. Vitamin B3 was fully recovered when spiked into the sample at its labeled value.44 Water-Soluble Vitamin 7 Detection: UV absorbance at 210 nm Valve Position: See Table 3 Samples: A_H. Table 5 reports the data from the calibration as calculated by Chromeleon CDS software.9994 1.26 VB12 245 0. VB12 50 7.0–100 A=0.

pp 1592–1605. Sunnyvale. 3. V. Sunnyvale. DC. As shown in Figure 7. 870. 207–215. 2013). [Online] www. K.. Sunnyvale. VC is a major peak in the sample chromatogram. Dionex (now part of Thermo Scientific) Application Note 216: Determination of Water. VB1 2. 1 mg/mL will be detected as 1 µg/mL. 24. 2. Yasmin.30 115 Detected — — — ND ND 1. Denmark +45 70 23 62 60 Europe Other +43 1 333 50 34 0 Finland +358 9 3291 0200 France +33 1 60 92 48 00 Germany +49 6103 408 1014 India +91 22 6742 9494 Italy +39 02 950 591 Japan +81 6 6885 1213 Korea +82 2 3420 8600 Latin America +1 561 688 8700 Middle East +43 1 333 50 34 0 Netherlands +31 76 579 55 55 New Zealand +64 9 980 6700 Norway +46 8 556 468 00 Thermo Fisher Scientific.1 1 2. products. Quantitative Simultaneous Estimation of Water Soluble Vitamins. A 2000. 24. 5. 4. com/en-us/webdocs/87181-AN251-HPLC-vitaminsupplements-25June2010-LPN2533. 2013). All rights reserved.com/en-us/ webdocs/113854-AN1023-LC-2D-Sudan-Dyes-CurryPaste-AN70212_E. 24. Sunnyvale. Dionex (now part of Thermo Scientific) Application Note 251: Determination of Water.03 6 25 30 Figure 7. The Open Analytical Chemistry Journal 2009. ND = not detected. A. P.dionex.dionex. [Online] www. Sunnyvale. Analysis of this complex sample requires only off-line filtration because the remainder of the sample preparation is automated by the UltiMate 3000 x2 Dual HPLC system and Chromeleon CDS software.01 VC mAU 4 1 0 5 5 10 15 Minutes 20 35 Detected (µg/mL) Added (µg/mL) 1. 8. VB5 5. The U. J. CA.pdf (accessed Oct. Khan. Sunnyvale.34 103 ND 2 2. and its subsidiaries. Africa +43 1 333 50 34 0 Australia +61 3 9757 4300 Austria +43 810 282 206 Belgium +32 53 73 42 41 Brazil +55 11 3731 5140 Canada +1 800 530 8447 China 800 810 5118 (free call domestic) 400 650 5118 Found (µg/mL) *The sample is diluted 1000 times before analysis. www. Dionex (now part of Thermo Scientific) Application Note 287: Two-Dimensional HPLC Combined with On-Line SPE for Determination of Sudan Dyes I–IV in Chili Oil. Thermo Scientific Application Note 1023: Determination of Sudan Dyes I–IV in Curry Paste. Unknown 6. CA USA is ISO 9001:2008 Certified. . 2008. Not all products are available in all countries. Specifications. 24. This 2D-HPLC method greatly simplifies the second-dimension chromatogram. Salvado. [Online] www. VB2 Analyte Labeled (mg/mL)* VB1 1 VB3 1 VB6 1 VB5 5 VB12 0.and Fat-Soluble Vitamins in Multivitamin Pharmaceutical Formulations by High-Performance Liquid Chromatography.015 VB2 1 VB7 0.dionex.3 1.com/dionex ©2013 Thermo Fisher Scientific Inc.dionex. Perveen. Conclusion Two-dimensional HPLC simplifies the determination of the vitamin content of a multivitamin nutritional drink. Determination of Eight Water.2 110 4. References 1.96 96 1. 24. All other trademarks are the property of Thermo Fisher Scientific Inc.. VB6 4. 3. 2011. 6. Russia/CIS +43 1 333 50 34 0 Singapore +65 6289 1190 Sweden +46 8 556 468 00 Switzerland +41 61 716 77 00 Taiwan +886 2 8751 6655 UK/Ireland +44 1442 233555 USA +1 800 532 4752 AN70755_E 01/14S Ap plica t ion Note 1 0 69 Although VC in the working standard solutions is quite unstable.and Fat-Soluble Vitamins in Functional Waters by HPLC with UV-PDA Detection. Vitamins/Dietary Supplements. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc.M. its presence in the multivitamin nutritional drink appeared to be relatively stable. 2013). terms and pricing are subject to change. com/en-us/webdocs/111142-AN287-HPLC-SudanDyes-Chili-Oil-21Sept2011-LPN2919. Please consult your local sales representative for details. Riboflavin. Cyanocobalamin and Folic Acid in Nutraceutical Products by HPLC. 3 –10 Table 6.98 95 1 0.03 76 1 0.and Fat-Soluble Vitamins in Nutritional Supplements by HPLC with UV Detection.51 2 6. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.pdf (accessed Oct. VC 2 3. Second dimension of multivitamin nutritional drink sample (100-fold dilution). Pharmacopeia 35. Chromatogr. CA. thus. Washington. 1–5. com/en-us/webdocs/71938-AN216-HPLC-VitaminsFunctionalWaters-30April09-LPN2145. 2010. ISO is a trademark of the International Standards Organization. as shown in Figure 7.Detection: UV absorbance at 245 nm Valve Position: See Table 3 Sample: Multivitamin nutritional drink sample Peaks: 250 1.S. [Online] www. Dionex (now part of Thermo Scientific) Technical Note 89: Determination of Water.03 Recovery (%) 1 1. [Online] www. Analysis results of water-soluble vitamins in the multivitamin nutritional drink sample. Moreno. CA.pdf (accessed Oct. Pyridoxine. 7. CA.pdf (accessed Oct. 2013).pdf (accessed Oct. 2013). 2010.and Fat-Soluble Vitamins by HPLC. a complex sample. NF 30. S..com/en-us/webdocs/88784-TN89-HPLCWaterFatSolubleVitamins-27Oct2010-LPN2598.thermoscientific.dionex. 2009.83 83 0. Good recoveries of water-soluble vitamins in the spiked sample (Table 6) provided another positive indicator of method accuracy. CA. 2012.